U.S. patent number 4,499,188 [Application Number 06/375,098] was granted by the patent office on 1985-02-12 for bacterial production of heterologous polypeptides under the control of a repressible promoter-operator.
This patent grant is currently assigned to Cetus Corporation. Invention is credited to Michael W. Konrad, David F. Mark.
United States Patent |
4,499,188 |
Konrad , et al. |
February 12, 1985 |
Bacterial production of heterologous polypeptides under the control
of a repressible promoter-operator
Abstract
A process for bacterially producing heterologous polypeptides,
particularly those such as human IFN-.beta. that inhibit bacterial
growth, in which bacteria that have been transformed to express the
heterologous polypeptide under the control of a trp
promoter-operator are cultivated in a known volume of medium
containing an excess of a preferred carbon source such as glucose
and a predetermined amount of tryptophan that corresponds
approximately to the amount of tryptophan contained in the bacteria
in the volume of medium at a predetermined elevated cellular
density, whereby expression of the heterologous polypeptide is
substantially repressed until the bacteria grow to approximately
the predetermined elevated cellular density and is thereafter
automatically derepressed to permit expression of the heterologous
polypeptide.
Inventors: |
Konrad; Michael W. (Alameda,
CA), Mark; David F. (Hercules, CA) |
Assignee: |
Cetus Corporation (Emeryville,
CA)
|
Family
ID: |
23479488 |
Appl.
No.: |
06/375,098 |
Filed: |
May 5, 1982 |
Current U.S.
Class: |
435/69.51;
930/10; 930/142 |
Current CPC
Class: |
C07K
14/565 (20130101); C12N 15/71 (20130101); Y10S
930/142 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/565 (20060101); C12N
15/71 (20060101); C12P 021/02 (); C12P 021/00 ();
C12N 015/00 () |
Field of
Search: |
;435/68-70,172,172.3
;935/41 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4374927 |
February 1983 |
Sninsky et al. |
|
Foreign Patent Documents
Other References
Mandelstam et al., Biochemistry of Bacterial Growth, John Wiley
& Sons, pp. 159 & 160, (1982). .
Derynck et al., Nature, vol. 285, pp. 542-546, Jun. 1980. .
Taniguchi et al., Nature, vol. 285, pp. 546-549, Jun. 1980. .
Edge et al., Nature, vol. 292, pp. 756-762, Aug. 1981. .
Crawford et al., Ann. Rev. Biochem., vol. 49, pp. 163-195
(1980)..
|
Primary Examiner: Tanenholtz; Alvin E.
Attorney, Agent or Firm: Halluin; Albert P. Hasak; Janet E.
Ciotti; Thomas E.
Claims
We claim:
1. A process for bacterially producing a heterologous polypeptide
comprising cultivating bacteria transformed to express said
heterologous polypeptide under the control of a bacterial
promoteroperator that
(i) normally controls the expression of the structural genes that
encode the polypeptides of a biosynthetic pathway that produces a
product that is used by the bacteria in growth,
(ii) is repressed by said product through activation of a repressor
molecule by said product, and
(iii) provides efficient expression of the structural genes under
its control when derepressed
in a volume of a culture medium containing an excess of essential
nutrients and a predetermined amount of said product that
corresponds approximately to the amount of said product contained
in the bacteria in said volume at a predetermined, elevated
cellular density for a time sufficient to permit growth of the
bacteria to the predetermined cellular density and expression of
the heterologous polypeptide thereafter.
2. The process of claim 1 wherein the promoter-operator is the trp
promoter-operator and the product is tryptophan.
3. The process of claim 1 or 2 wherein the heterologous polypeptide
affects bacterial growth or viability adversely.
4. The process of claim 1 or 2 wherein the heterologous polypeptide
is human IFN-.beta..
5. The process of claim 1 or 2 wherein the heterologous polypeptide
is human IFN-.beta., the bacteria are E.coli, and the trp
promoter-operator is the E.coli trp promoter-operator.
6. The process of claim 5 wherein the predetermined amount of
tryptophan is defined by the formula:
7. The process of claim 1 or 2 wherein the predetermined elevated
cellular density is substantially maximum cellular density.
8. The process of claim 2 wherein the predetermined elevated
cellular density as measured by the turbidity of the medium is in
the range of about 10 to about 100.
9. The process of claim 8 wherein the bacteria are E.coli and the
predetermined amount of tryptophan is in the corresponding range of
about 53 mg/l of medium to about 530 mg/l of medium.
10. The process of claim 9 wherein the bacteria have been
transformed with the plasmid p.beta.l-trp.
Description
DESCRIPTION
1. Technical Field
The invention is in the field of genetic engineering. More
particularly it concerns a process for making heterologous
polypeptides by cultivating certain genetically engineered bacteria
under certain culture conditions.
2. Background Art
Various bacterial expression control DNA sequences have been used
to control the expression of foreign (heterologous) polypeptides by
transformed bacteria. One of these is the sequence that controls
the expression of the structural genes of the tryptophan (trp)
operon. Goeddel, et al, Nucl Acid Res (1980) 8:4057-4074, describe
the construction of chimeric plasmids containing a trp control
sequence linked to the IFN-.beta. structural gene. E.coli
transformed with these plasmids are reported to produce human
IFN-.beta.. Edman, et al, Nature (1981) 291:503-506, describe the
construction of chimeric plasmids containing a portion of the trp
control sequence linked to the structural genes that encode the
Hepatitis B core antigen or a .beta.-lactamase:Hepatitis B surface
antigen fusion polypeptide. Expression of both of these
polypeptides by transformed E.coli is reported.
European patent application No. 36776, published Sept. 30, 1981,
describes plasmids containing a bacterial trp promoter-operator
sequence linked to structural genes that encode somatostatin, human
growth hormone, thymosin .alpha. 1, or polypeptides containing
those heterologous polypeptides fused to a bacterial polypeptide.
The plasmids lack a trp attenuator region. E.coli are transformed
with these plasmids and transformants are grown in a medium to
which tryptophan is added to repress the trp operator. Once the
recombinant culture has grown to a level appropriate for industrial
production of the foreign polypeptide the external source of
tryptophan is removed, thereby derepressing the trp operator to
allow expression of the foreign polypeptide. Only one technique for
removing tryptophan from the medium is described. It involves
diluting the tryptophan-rich medium in which the transformants are
grown into a large volume of a medium containing no additive
tryptophan. This dilution technique, while perhaps feasible on a
laboratory scale, has significant disadvantages if practiced in a
large scale cultivation. Among these disadvantages is a requirement
either to measure the amount of tryptophan in the medium at the
time of dilution in order to know how much to dilute or dilute
many-fold and the necessity for additional equipment to add the
diluent medium.
A main object of the invention is to provide a process for
producing a heterologous polypeptide by the expression in bacteria
of a structural gene coding for said polypeptide under the control
of a repressible bacterial promoter-operator in which the bacteria
are grown in a manner in which the culture medium is automatically
depleted of additive repressor at a predetermined cellular density.
This unique process requires no monitoring of repressor levels in
the culture medium and no addition of diluent medium.
DISCLOSURE OF THE INVENTION
The invention is a process for bacterially producing a heterologous
polypeptide comprising cultivating bacteria transformed to express
said heterologous polypeptide under the control of a bacterial
promoter-operator that
(i) normally controls the expression of the structural genes that
encode the polypeptides of a biosynthetic pathway that produces a
product that is used by the bacteria in growth,
(ii) is repressed by said product through activation of a repressor
molecule by said product, and
(iii) provides efficient expression of the structural genes under
its control when derepressed
in a predetermined volume of a culture medium containing an excess
of essential nutrients and a predetermined amount of said product
that corresponds approximately to the amount of said product that
would be theoretically contained in the bacteria in said volume at
a predetermined cellular density for a time sufficient to permit
growth of the bacteria to said predetermined cellular density and
expression of the heterologous polypeptide thereafter. Using this
process the expression of the heterologous polypeptide is
substantially repressed until the cellular density reaches
approximately the predetermined cellular density and is thereafter
derepressed automatically without external intervention to permit
expression of the heterologous polypeptide.
This process is particularly advantageous for producing
heterologous polypeptides, such as human IFN-.beta., that affect
bacterial growth or viability adversely.
DESCRIPTION OF THE DRAWINGS
In the drawings:
FIG. 1 is a restriction map of the human IFN-.beta. cDNA clone
described in Example 1, infra, and includes a diagram of the
strategy used to determine the DNA sequence of that clone;
FIG. 2 is the DNA sequence of the coding strand of the coding
region of the IFN-.beta. cDNA clone described in Example 1, infra,
and the amino acid sequence corresponding thereto;
FIG. 3 depicts the subcloning of the IFN-.beta. gene coding
sequence described in Example 1.
FIG. 4 is a restriction map of the plasmid p.beta.1-trp described
in Example 1, infra;
FIG. 5 is the DNA sequence between the EcoRI site and the XhoII
site of p.beta.1-trp as shown in the enlargement of FIG. 4 and the
amino acid sequence coresponding thereto; and
FIG. 6 is a graph of optical density versus time for the
cultivations described in Example 3, infra.
MODES FOR CARRYING OUT THE INVENTION
As used herein the term "heterologous" refers to a structural gene
not found in wild type bacteria and to polypeptide sequences not
produced by such bacteria. Heterologous genes and polypeptides are
typically eukaryotic genes and polypeptides.
As used herein the term "promoter-operator" denotes a sequence,
native or engineered, of nucleotides that controls and regulates
the expression of the linked structural genes that encode the
polypeptides of a biosynthetic pathway. The nucleotide sequence
begins with the promoter, includes the operator, the transcription
start site, and the Shine-Delgarno sequence, and ends with the
start codon for the expression of those polypeptides.
As used herein the term "structural gene" denotes that part of the
relevant gene that encodes the amino acid sequence(s) for the
expressed polypeptides under the control of the
promoter-operator.
As used herein the term "expression" denotes the process by which a
structural gene produces a polypeptide. It involves transcription
of the relevant gene into messenger RNA (mRNA) and the translation
of such mRNA into a polypeptide.
As used herein the term "vector" denotes the recombinant DNA
molecules that include a heterologous structural gene under the
control of a promoter-operator and: (1) are able to replicate
autonomously in host bacteria, and (2) contain a marker function
that allows selection of host cells transformed by the vector. This
term is synonomous with the art term "cloning vehicle".
As used herein the term "transformed bacteria" denotes host
bacteria that have been genetically engineered to produce a
heterologous polypeptide under the control of a promoter-operator.
Such bacteria are sometimes referred to herein as
"transformants".
As used herein the term "repression" denotes the inactivation of
the promoter such that the expression of structural genes
controlled thereby is substantially inhibited.
As used herein the term "IFN" is synonomous with the term
"interferon". Correspondingly, the terms "IFN-.alpha." and
"IFN-.beta." are synonomous with the terms "leukocyte interferon"
and "fibroblast interferon", respectively.
As used herein the term "human IFN-.beta." denotes IFN-.beta. that
is produced by transformants that have been transformed with a
vector that includes a human structural IFN-.beta. gene or a human
structural IFN-.beta. gene that expresses an IFN-.beta. whose amino
acid sequence is the same as or substantially homologous to native
human IFN-.beta. under the control of a bacterial trp
promoter-operator.
As used herein the term "turbidity" denotes cellular density as
measured by optical density at 680 nm using a spectrophotometer or
colorimeter.
The heterologous polypeptides that are made by the invention
process will typically have industrial, agricultural, or medical
utility. In most instances the polypeptides will be nonbacterial
proteins such an eukaryotic cell proteins and viral proteins.
Biologically active vertebrate cell proteins, especially human cell
proteins, such as hormones, immunoregulatory molecules, antigens,
and antibodies are of particular interest.
As indicated above the heterologous polypeptide is expressed under
the control of a bacterial promoter-operator that normally controls
the expression of a biosynthetic pathway that makes a product that
is used by the bacteria in their growth. Such promoter-operators
include the trp promoter-operator, the tyrA promoter-operator, the
tyrB promoter-operator, and the pheA promoter-operator. These
promoter-operators are repressed, respectively, by interaction of
tryptophan, tyrosine, and phenyl alanine with their respective
repressor proteins, the complex then binds to the respective
operator and inactivates the promoter. The trp promoter-operator is
preferred and, for convenience, the following description relates
to the embodiment of the invention using that promoter-operator and
tryptophan as a repressor.
The transformed bacteria that are employed in the invention process
may be engineered using the procedures described by Goeddel, et al,
supra, Edman, et al, supra, and European patent application Ser.
No. 36776. These procedures basically involve (i) synthesizing or
isolating the desired structural gene sequence that encodes the
heterologous polypeptide, (ii) cloning the DNA sequence into an
appropriate plasmid or viral vector at a site that is controlled by
a bacterial trp promoter-operator, a ribosome binding site for the
translation of the transcribed mRNA, and a translation start codon
in the same translational reading frame as the structural gene,
(iii) introducing the cloned sequence into competent bacterial
cells, and (iv) selecting transformants either by a plasmid marker
function or their ability to produce the heterologous polypeptide.
E.coli are preferred for use in the process.
The heterologous polypeptide-producing transformants are introduced
into a known volume of a culture medium that contains a
predetermined amount of tryptophan that corresponds to the amount
of tryptophan that would be theoretically contained in the bacteria
in said volume at a predetermined cellular density. In addition to
the added tryptophan, the medium will contain an excess of nutrient
materials (other than tryptophan) that fulfill the cellular growth
requirements of the bacteria thereby enabling the bacteria to grow
and multiply to the predetermined cellular density. Such materials
will include sources of carbon and nitrogen for synthesis of
cellular components and energy, minerals (ions) such as sulfur
(SO.sub.4.sup.-2), phosphorous (PO.sub.4.sup.-3), Mg.sup.+2,
K.sup.+, and Ca.sup.+2, amino acids, purines, pyrimidines, and
vitamins. Trace elements will usually be contained in the water
source for undefined media but must be added to defined media.
Oxygen for facultative and aerobic bacteria will also be provided
to the medium. In order to achieve maximum cellular densities, the
cultivation will usually be carried out in a manner that enhances
the area of the oxygen-liquid interface.
Important environmental factors affecting the cultivation include
pH and temperature. The temperature will range between the minimal
and maximum growth temperatures. Most bacteria exhibit maximum
growth over a fairly narrow temperature range. For mesophilic
bacteria, such as E.coli, the optimum temperature range is about
30.degree. C. to about 40.degree. C. Most organisms will tolerate
hydrogen ion concentration ranging over several pH units. For
pathogenic bacteria, such as E.coli, the tolerable pH lies in the
range of about 6 to 8, with 6.5 being preferred.
Tryptophan is added to the medium before the cultivation is
initiated in an amount that is correlated to the volume of culture
medium and the approximate amount of tryptophan that would
theoretically be in the cell mass in the volume at a predetermined
cellular density. In the presence of excess preferred carbon
source, such as glucose, the bacteria will use the tryptophan in
the medium rather than producing it themselves for use in the
production of cellular protein. While added tryptophan is present
the bacteria repress expression of the heterologous polypeptide
under the control of the trp promoter-operator. By initially adding
a proper amount of tryptophan to the media the bacteria may be
grown to a predetermined cellular density with the trp operator
repressed. In order to determine this amount the volume of the
medium must be known or determined, the amount of tryptophan per
unit dry weight of the bacterial protein is determined
experimentally or from published sources (for E.coli see Studies of
Biosynthesis in Eschericia Coli, Roberts, et al, Carnegie
Institution of Washington Publication 607 (1955), p28), and a
desired cellular density is selected.
Cellular density may be expressed as grams (dry weight) of cells
per unit volume of medium or in terms of the turbidity of the
medium as measured with a photoelectric colorimeter (e.g. a Klett
turbidometer) or spectrophotometer. Since turbidity is the most
convenient measure of cellular density it is desirable to define
the relationship between tryptophan addition and cellular density
in terms of turbidity. For E.coli the relationship between
turbidity and the grams (dry wt) of cells per unit volume of medium
is linear, with one turbidity unit being approximately equal to
0.48 g (dry wt) cells per liter of medium. Using this relationship
and the amount of tryptophan per unit weight (dry basis) of cells
(approximately 11 mg tryptophan per g of cells for E.coli) the
amount of tryptophan to be initially added to the medium per
turbidity unit may be calculated. For E.coli that amount is:
While the bacteria may be grown to less than maximum density, they
will usually be grown to substantially maximum cellular density,
e.g. the density at which oxygen transfer becomes limiting. Such
densities are normally characterized by turbidities in the range of
about 10 to 100. In the case of E.coli, the amount of tryptophan to
be initially added to the medium, determined from formula (1)
above, will correspondingly range between 53 to 530 mg/l. When the
turbidity of the medium reaches the predetermined turbidity, the
amount of tryptophan added to the medium will have been consumed by
the bacteria in producing cellular protein. The tryptophan is thus
depleted automatically by cell growth and there is no need to
intervene to dilute the medium or otherwise remove or monitor
tryptophan in the medium. Once the tryptophan has been so depleted,
the trp operator is derepressed and expression of the heterologous
polypeptide is initiated.
The same cultivation conditions may be used in the heterologous
polypeptide expression phase of the cultivation as were used in the
exponential cell growth phase of the cultivation. The only
exception to this is the absence of tryptophan in the culture
medium during the expression phase. During the expression phase the
growth rate will decrease if the heterologous polypeptide is one
that affects cell growth/viability adversely. If the heterologous
polypeptide does not so affect the cells, cell growth during the
expression phase will be similar to normal growth. The duration of
the expression phase may vary depending upon the bacteria,
heterologous polypeptide, and cultivation conditions. The
expression phase duration will usually be in the range of 1 to 5
hr.
After the expression phase the heterologous polypeptide is
recovered from the cells and/or culture medium. In the case of
IFN-.beta. production by E.coli, the cells are harvested and the
IFN-.beta. is recovered therefrom. A preferred process for
recovering human IFN-.beta. from bacteria is disclosed in commonly
owned, copending U.S. patent application Ser. No. 353,360 filed
Mar. 1, 1982, titled "Process for Recovering Human IFN-.beta. from
a Transformed Microorganism" now U.S. Pat. No. 4,450,103. That
recovery process involves disrupting the solubilizing the
IFN-.beta. into an aqueous medium with an anionic surfactant such
as sodium dodecyl sulfate or sodium laurate and extracting the
solubilized IFN-.beta. from the aqueous medium with 2-butanol,
2-methyl-2-butanol, or mixtures thereof.
The following examples further describe the materials and methods
used in carrying out the invention. The examples are not intended
to limit the invention in any manner.
EXAMPLE 1
Construction of plasmid p.beta.1-trp for the direct expression of
IFN-.beta. under, the control of trp promoter-operator
The human IFN-.beta. cDNA clone, 4El, was obtained by reverse
transcriptase synthesis of cDNA using oligo-dT as primer, and as
template, mRNA derived from human primary foreskin fibroblast
cells. The cDNA was made double-stranded by the action of E.coli
DNA polymerase I and nicked with Sl-nuclease. Homopolymeric tails
were added to the 3'-termini of the ds cDNA by the enzyme
terminal-transferase using dCTP as substrate. Similar homopolymeric
tails were added to the 3'-termini of the plasmid pBR322 which had
been linearized at the PstI site, using dGTP as substrate. The
plasmid pBR322 and the ds cDNA were hybridized and transformed into
E.coli K12. The clone, 4El, was identified by Grunstein-Hogness
(PNAS (1975) 72:3961) colony hybridization screens using a .sup.32
P-labeled probe. Further characterization of the clone by
restriction enzyme analysis gave the following results. PstI
digestion of 4El yielded two insert fragments of about 600 bp and
200 bp in addition to a fragment corresponding to linear pBR322
DNA. BglII-PstI digestion of the same clone showed that the 600 bp
PstI insert fragment can be further digested with BglII to yield
two fragments of sizes 358 bp and 250 bp. HinfI digests of clone
4El showed that there are at least three HinfI sites in the insert
fragment to generate three new fragments that are not present in
pBR322.
FIG. 1 is a restriction map of clone 4El that includes a diagram
showing the sequencing strategy used to sequence the coding region
of the clone. The DNA sequence was obtained by a combination of
Sanger's sequencing, (PNAS, (1977) 74:5463-5467) and Maxam-Gilbert
sequencing (PNAS, (1977) 74:560-564). The Sanger technique was used
to sequence fragment D2 whereas the remaining fragments (A, B, C,
and D1) were sequenced by the Maxam-Gilbert method. The DNA
sequence for the coding region of the IFN-.beta. clone, 4El, and
the corresponding predicted amino acid sequence are shown in FIG.
2.
The IFN-.beta. gene coding sequence was subcloned by using a
synthetic oligonucleotide primer (TATGAGCTACAAC) and the enzyme DNA
polymerase I to degrade DNA sequences 5' to the ATG codon which
codes for the amino-terminal methionine of the mature interferon,
thereby removing the DNA sequences coding for the leader peptide
(FIG. 3). The repaired DNA was then subcloned into pBR322 at the
repaired HindIII and at the BamHI sites. The BglII site in the
IFN-.beta. gene, just past the UGA termination codon, was used to
ligate with the BamHI cohesive end in pBR322 to generate an XhoII
site, while the repaired 5'-terminus of the IFN-.beta. gene was
blunt-end ligated to the repaired HindIII site; because the
original primer has an extra thymidine nucleotide at its
5'-terminus, the HindIII site was regenerated. The resulting clone,
p.beta.l-25, was analyzed by restriction analysis and DNA sequence
analysis to confirm the presence of the HindIII site and the
integrity of the initiation codon.
The E.coli trp promoter and ribosome binding site, which has
previously been subcloned into pBR322, was ligated into p.beta.l-25
as an EcoRI-HindIII fragment.
The resulting clone, p.beta.1-trp, was analyzed by restriction
enzymes. FIG. 4 is a restriction map of p.beta.1-trp that includes
an enlargement of the trp promoter operator region and the
IFN-.beta. coding sequence. FIG. 5 shows the nucleotide sequence
between the EcoRI site and the XhoII site of p.beta.1-trp as shown
in the enlargement of FIG. 4.
EXAMPLE 2
Effect of Tryptophan on growth of E.coli transformed with
p.beta.1-trp
E. coli were transformed with p.beta.1-trp by conventional
procedures (Mandell, M. and Higa, A. (1970) J Mol Biol 53:159) to
produce a transformant line identified as MM294.
Twenty-five ml samples of MM294 growing in the medium described
below were centrifuged at 10000.times.g for 10 min. The cells were
resuspended in minimal medium containing (1) no tryptophan and (2)
20 .gamma./ml tryptophan. Turbidity readings of each media were
taken at various time intervals spanning about a five and one-half
hour period. These turbidities are tabulated below.
______________________________________ Turbidity (Klett units)
Reading No No Tryptophan Tryptophan
______________________________________ 1 21 20 2 20 20 3 26 27 4 26
32 5 30 46 6 30 66 7 39 125
______________________________________
The culture medium comprised (concentrations indicated are
sufficient for growth to 15 g/l dry weight):
______________________________________ Base Medium Additives
Ingredient Concentration Ingredient Concentration
______________________________________ (NH.sub.4).sub.2 SO.sub.4 72
mM FeSO.sub.4.7H.sub.2 O 72 .mu.M KH.sub.2 PO.sub.4 21.6 mM
thiamine.HCl 20 mg/l MgSO.sub.4.7H.sub.2 O 3 mM ampicillin 20 mg/l
Na citrate.2H.sub.2 O 1.5 mM glucose 30-50 g/l ZnSO.sub.4.7H.sub.2
O 45 .mu.M MnSO.sub.4.4H.sub.2 O 45 .mu.M CuSO.sub.4.5H.sub.2 O 4.5
.mu.M ______________________________________
These turbidity data show that the induction of IFN-.beta. in the
culture that did not contain tryptophan had an adverse effect on
cell growth. In contrast, in the culture containing tryptophan the
induction of IFN-.beta. was repressed, thereby permitting the
bacteria to grow at a characteristic exponential rate.
EXAMPLE 3
Cultivation of MM 294 Using Varying Initial Tryptophan
Concentrations
MM 294 cell samples were grown and prepared as in Example 2. A
laboratory fermenter was inoculated at a low density with a seed
culture of these cells growing in minimal medium containing
tryptophan at 70 mg/l and 364 mg/l. Turbidity readings were made
periodically. FIG. 6 is a plot of logarithm of turbidity versus
time for the two fermentations.
Using formula (1), supra, it was predicted that an initial
tryptophan concentration of 70 mg/l would permit the cells to grow
to a turbidity of about 13.4 before the tryptophan would be
exhausted and the trp operator would be derepressed. It was
similarly predicted that at 364 mg/l the turbidity at tryptophan
exhaustion would be about 69. At these turbidities it was expected
that cell growth would begin to be inhibited which would be
reflected as breaks in the growth curves for the cultivations. As
shown in FIG. 6 the observed breaks in the curves appear at about a
turbidity of 12 for the 70 mg/l cultivation and at about 60 for the
364 mg/l cultivation. These observed values correspond reasonably
well to the predicted values.
A similar cultivation of E.coli transformed to directly express
IFN-.alpha. under control of the E.coli trp promoter-operator in a
culture medium containing 25 mg tryptophan per 1 of medium showed
no slowing of cellular growth through a turbidity of 20. Tryptophan
was theoretically exhausted from the medium at a turbidity of about
4.7. These results indicate that IFN-.alpha., unlike IFN-.beta.,
has no apparent adverse affect on E.coli growth and that the use of
added tryptophan in such cultivations provides no advantageous
results.
Modifications of the above described modes for carrying out the
invention that are obvious to those of ordinary skill in the
biochemical engineering field or related technologies are intended
to be within the scope of the following claims.
* * * * *